[Prokaryotic expression and CRT-binding activity analysis of Hevea HbCBF1 protein]

CBF pathway is the most important pathway during the process of cold acclimation in plants. In previous study, we cloned a CBF like gene HbCBF1 from Hevea brasiliensis, which implied that CBF pathway might exist in this kind of tropical oriented tree. However, it was still unknown if this gene functioned as C-repeat binding factor. Therefore it was very important to analyze the C-repeat binding activity of HbCBF1. Using a prokaryotic expression system, the HbCBF1 was expressed and the soluble fusion protein was extracted under the optimized conditions. The C-repeat binding activity of HbCBF1 protein was analyzed by gel shift assay, which indicated that the fusion protein could bind to the COR15a probe efficiently, and this binding activity could be com-peted with by overloading non-labeled COR15a probe. An unlabeled mutated probe M1, which did not include the C-repeat sequence, could not compete with the binding activity of HbCBF1 to COR15a probe. These results indicated HbCBF1 fusion protein could bind to the C-repeat containing DNA fragment specifically in vitro.